Methods for harvesting microalgae

ABSTRACT

Methods for harvesting microalgae, comprise providing water containing microalgae, providing flocculant in an amount to form flocs of microalgae when mixed with the water, generating a mixture of the water and the flocculant and separating the flocs from the mixture.

CROSS-REFERENCE(S) TO RELATED APPLICATION(S)

Not applicable

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

Not applicable

PARTIES TO A JOINT RESEARCH AGREEMENT

Not applicable

REFERENCE TO A SEQUENCE LISTING SUBMITTED ON A COMPACT DISC

Not applicable

BACKGROUND

The present disclosure relates generally to algaculture, and morespecifically to culturing and harvesting microalgae.

Microalgae are unicellular, microscopic algae that produce variouscarbohydrates, proteins, and lipids useful for numerous industrial,nutritional, and medical/pharmaceutical applications. Microalgae are animportant starting material for the production of biofuels, nutritionalsupplements, and pharmaceuticals. (Available at:www.rsisinternational.org/Issue7/313-317.pdf) A cost-efficient,high-yield and reliable means of producing the algal biomass for theseapplications is critical to the sustainability of algaculture andproduction of the above-mentioned materials.

Microalgae are photosynthetic organisms capable of growing in diverseaquatic conditions and can be found or cultured in freshwater,saltwater, and brackish water. While microalgae can thrive in a varietyof conditions, cultivation conditions impact their growth rate as wellas their lipid, protein, and carbohydrate content. For enhanced growthof the microalgae and optimal protein/carbohydrate/lipid content of themicroalgae, specific parameters with respect to nutrient quantity andquality, light, pH, turbulence, salinity and temperature must becarefully maintained. (Available at:www.fao.org/docrep/003/w3732e/w3732e06.htm)

Microalgae can be cultivated both indoors and outdoors, in ponds,raceways or closed containers. Isolation of the microalgae from theliquid culture requires separation and concentration of the microalgaefrom large amounts of water. This can be accomplished through variousmeans. Flocculation and coagulation cause the single-celled microalgaeto clump and serve to increase the particle size of the microalgae tofacilitate harvesting. While this step is not always necessary,microalgae clumps or flocs can then be more easily separated from thewater through centrifugation, floatation or filtering. (Available at:www.rsisinternational.org/Issue7/313-317.pdf and atwww.fao.org/docrep/003/w3732e/w3732e06.htm)

Microalgae used to produce extracts for human consumption requireespecially stringent culturing and harvesting standards. Particularlywhen grown outdoors in open systems, microalgae can be prone tocontamination by other algal species or opportunistic, microbialorganisms such as fungi, molds and bacteria. Furthermore, chemical orforeign material contamination whether from outside sources or from themethod itself (e.g., chemical flocculants) also should be carefullymonitored and steps taken to minimize such potential sources ofcontamination.

Further to these considerations, any method for microalgae cultivationand harvesting must also account for its long-term sustainability andeconomic feasibility. The cultivation and harvesting of high-qualitymicroalgae for use, and particularly for producing extracts for humanconsumption, requires extensive resource investment. There exists a needin the art for an efficient, cost-effective, and reliable method forcultivation and harvesting of microalgae for use towards the productionof algal extracts and nutraceuticals.

BRIEF SUMMARY

The presently claimed method provides a reliable and efficient means ofculturing and harvesting microalgae. The method addresses the specificneeds of providing cultivation and harvesting conditions that promoteoptimal growth of the microalgae species of interest and enhance thelipid, protein, and carbohydrate content/composition for application inproduction of algal extracts and nutraceuticals.

The presently claimed method provides a cost-effective means ofharvesting microalgae free from harmful contaminants, such as chemicalflocculants, microbial contaminants, or environmental impurities. Themethod therefore is useful for generating algal biomass that can beprocessed into microalgae-derived nutraceuticals and other manufactures.The method is cost-effective as it directly utilizes sea water,advantageously repurposes flue gas, and economically recycles the waterfor re-use in microalgae cultivation. Furthermore, costs are greatlyreduced by cultivating microalgae in open vessels whereby oxygen neednot be specially provided to support growth and also by flocculation ofthe microalgae prior to harvesting to more efficiently separate waterfrom the microalgae to allow harvesting. The microalgae provided in theclaimed method are capable of producing eicosapentaenoic acid (EPA)across a broad range of temperatures permitting for outdoor culture. Inview of these factors, the presently claimed method offers acost-effective and efficient means of cultivating, isolating andharvesting microalgae by harnessing the benefits of the above-namedfactors to generate superior quality microalgae at high-yield.

The embodiments described herein meets the needs described in the abovesection by providing methods for harvesting microalgae by providingwater containing microalgae, generating a mixture of the water andflocculant, and separating the flocs from the mixture. In certainembodiments, the flocculant is chitosan. In some embodiments of theabove-named aspects, chitosan is provided at a concentration of about 2%of the weight of the microalgae in the mixture.

In some embodiments, the microalgae can produce eicosapentaenoic acid(EPA) at temperatures above 32° C. In certain embodiments, themicroalgae is Nannochloropsis.

In certain embodiments, the water has pH between about 8.0 and about9.0. In some embodiments, the water has salinity of about 2.2%. Incertain embodiments, the water has sodium hydrogen carbonate at aconcentration between about 0.1 g/L and about 0.2 g/L. In someembodiments, the water is at a temperature between about 10° C. andabout 38° C.

In certain embodiments, the method further comprises adding flue gas ormixing the water with flue gas. In some embodiments, the flue gas hasbetween about 10% and about 14% carbon dioxide. In some embodiments, theflue gas has less than 5 ppm of sulfur dioxide. In certain embodiments,the mixture of the water and flocculant is acidic. In some embodiments,the mixture has a pH of about 5.5.

Certain aspects of the present disclosure relate to methods of preparinga crude microalgal extract. Other aspects of the present disclosurerelate to methods of preparing a microalgal lipid extract.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the setup of air pump and filtration design.

FIG. 2 demonstrates the photo-bioreactor and light panel setup as partof the scale-up process.

FIG. 3 depicts construction of the outdoor ponds for microalgaecultivation. The ponds are fully lined with HDPE film.

FIG. 4 illustrates the design of the outdoor raceway, including thesiding, paddle wheel, and motor.

FIG. 5 depicts a flow-chart diagramming the method.

DETAILED DESCRIPTION

The following description sets forth exemplary methods, parameters andthe like. It should be recognized, however, that such description is notintended as a limitation on the scope of the present disclosure but isinstead provided as a description of exemplary embodiments.

Definitions

The term “flocculant” as used herein refers to an agent that causesaggregation of particles. Flocculant can describe an agent that causesclumping of microalgae to form a microalgae slurry or flocs.

The term “microalgae” as used herein refers to microscopic, unicellularalgae.

The term “flue gas” as used herein refers to gas that is released from aflue. Flue gas includes exhaust gas produced by power plants orfactories.

Methods of Harvesting Microalgae

The present disclosure relates to a method of harvesting microalgae byproviding water containing microalgae, providing an amount of flocculantsufficient to form flocs of microalgae when mixed with water, generatinga mixture of the water and flocculant, wherein the mixture is acidic,and separating the flocs from the mixture, thereby harvesting themicroalgae.

In certain embodiments, the water is saltwater, freshwater, or brackishwater. In other embodiments, the water is seawater. In a preferredembodiment, the water is pumped from the ocean.

In certain embodiments, the water has a pH between about 6.0 and about10.0. In other embodiments, the water has a pH between about 6.0 andabout 6.5, about 6.5 and about 7.0, about 7.0 and about 7.5, about 7.5and about 8.0, about 8.0 and about 8.5, about 8.5 and about 9.0, about9.0 and about 9.5, or about 9.5 and about 10.0. In certain embodiments,the water has a pH of about 6.0, about 6.5, about 7.0, about 7.5, about8.0, about 8.5, about 9.0, about 9.5, or about 10.0. In a preferredembodiment, the water has a pH between about 8.0 and about 9.0.

In certain embodiments, the water has a salinity between about 0.01% andabout 5%. In other embodiments, the water has a salinity between about0.01% and about 0.5%, about 0.5% and about 1.0%, about 1.0% and about1.5%, about 1.5% and about 2.0%, about 2.0% and about 2.5%, about 2.5%and about 3.0%, about 3.0% and about 3.5%, about 3.5% and about 4.0%,about 4.0% and about 4.5%, or about 4.5% and about 5.0%. In a preferredembodiment, the water has a salinity of about 2.2%.

In certain embodiments, the water comprises sodium hydrogen carbonate ata concentration between about 0.01 g/L and about 0.5 g/L. In someembodiments, the water comprises sodium hydrogen carbonate at aconcentration between about 0.01 g/L and about 0.05 g/L, about 0.5 g/Land about 1.0 g/L, about 1.0 g/L and about 1.5 g/L, about 1.5 g/L andabout 2.0 g/L, about 2.0 g/L and about 2.5 g/L, about 2.5 g/L and about3.0 g/L, about 3.0 g/L and about 3.5 g/L, about 3.5 g/L and about 4.0g/L, about 4.0 g/L and about 4.5 g/L, or about 4.5 g/L and about 5.0g/L. In a preferred embodiment, the water comprises sodium hydrogencarbonate at a concentration between about 0.1 g/L and about 0.2 g/L.

In certain embodiments, the flocculant is a polysaccharide that formscationic polyelectrolytes to precipitate the microalgae. In someembodiments, the flocculant is non-toxic. In other embodiments, theflocculant is derived from crustaceans. In yet other embodiments, theflocculant is chitosan. In certain embodiments, the chitosan is providedat a concentration between about 0.08% and about 3.5% of the weight ofthe microalgae in the mixture. In other embodiments, the chitosan isprovided at a concentration between about 0.08% and about 0.5%, about0.5% and about 1.0%, about 1.0% and about 1.5%, about 1.5% and about2.0%, about 2.0% and about 2.5%, about 2.5% and about 3.0%, or about3.0% and about 3.5% of the weight of the microalgae in the mixture. In apreferred embodiment, the chitosan is provided at about 2% of the weightof the microalgae in the mixture.

In certain embodiments, the method comprises adding flue gas or thewater is mixed with flue gas. In certain embodiments, the flue gascomprises carbon dioxide. In certain embodiments, the flue gas comprisescarbon dioxide at a concentration between about 4% and about 20%. Incertain embodiments, the flue gas comprises carbon dioxide at aconcentration between about 4% and about 8%, about 8% and about 12%,about 12% and about 16%, or about 16% and about 20%. In otherembodiments, the flue gas comprises carbon dioxide at a concentrationbetween about 6% and about 10%, about 10% and about 14%, or about 14%and about 18%. In a preferred embodiment, the flue gas comprises carbondioxide at a concentration between about 10% and about 14%. In certainembodiments, the flue gas has less than 10 ppm, 9 ppm, 8 ppm, 7 ppm, 6ppm, 5 ppm, 4 ppm, 3 ppm, 2 ppm, or 1 ppm of sulfur dioxide. In apreferred embodiment, the flue gas has less than 5 ppm of sulfur oxide.

In certain embodiments, the mixture is acidic. In certain embodiments,the mixture has a pH between about 4.5 and about 6.5. In someembodiments, the mixture has a pH between about 4.5 and about 5.0, about5.0 and about 5.5, about 5.5 and about 6.0, or about 6.0 and about 6.5.In a preferred embodiment, the mixture has a pH of about 5.5.

In certain embodiments, the water containing the microalgae is at atemperature between about 10° C. and about 38° C. In other embodiments,the water is at a temperature between about 10° C. and about 15° C.,about 15° C. and about 20° C., about 20° C. and about 25° C., about 25°C. and about 30° C., about 30° C. and about 35° C., or about 35° C. andabout 38° C.

In certain embodiments, after most of the water is drained for recycleuse, 15% of the flocs plus remaining water is left in the harvest ponds.In other embodiments, 10% of the flocs plus remaining water or 5% of theflocs plus remaining water is left in the harvest ponds. In a preferredembodiment, 2% of the flocs plus remaining water is left in the harvestponds.

In certain embodiments, centrifuging the flocs and remaining water is ata separation coefficient between about 1800 and about 3500. In apreferred embodiment, centrifuging the flocs and remaining water is at aseparation coefficient of about 2100. In some embodiments, the flocscontain about 10% dry algae weight (i.e., 100 g/L). In otherembodiments, the flocs contain 1-2% dry algae weight (i.e., 10 g-20 g/L)or less than 1% dry algae weight (i.e., less than 10 g/L).

In certain embodiments, the microalgae has a high oil content (>20%). Inother embodiments, the microalgae comprises 10%, 15%, 20%, 25%, 30%, or35% polyunsaturated fatty acids. In certain embodiments, the microalgaecan produce eicosapentaenoic acid (EPA) at temperatures above 32° C. Incertain embodiments, the microalgae canproduce >1%, >2%, >3%, >4%, >5%, >6%, >7%, >8%, >9%, >10%, >11%, >12%, >13%, >14%,or >15% EPA.

In certain embodiments, the microalgae is a marine microalgae. In oneaspect of the above-named embodiment, the microalgae can grow inseawater provided directly from the ocean. In other embodiments, themicroalgae is a freshwater or brackish water microalgae. In otherembodiments, the microalgae exist as single cells, as groups, or inchains. In other embodiments, the microalgae is Isochrysis sp.,Pseudoisochrysis sp., Dicrateria sp., Monochrysis sp., Tetraselmis sp.,Pyramimonas sp., Micromonas sp., Chroomonas sp., Cryptonmonas sp.,Rhodomonas sp., Chlamydomonas sp., Chlorococcum sp., Olisthodiscus sp.,Carteria sp., Dunaliella sp., Spirulina sp., Haematococcus sp., Rhodellasp., Arthrospira maxima, or Nannochloropsis sp. In a preferredembodiment, the microalgae is Nannochloropsis sp. In certainembodiments, the microalgae is Nannochloropsis gaditana, Nannochloropsisgranulate, Nannochloropsis limnetica, Nannochloropsis oceanica,Nannochloropsis oculata, or Nannochloropsis salina. In some embodiments,the Nannochloropsis may comprise genetic differences from wild-typeNannochloropsis or Nannochloropsis found in nature.

Other Methods of the Disclosure

The present disclosure also relates to a method of preparing a crudemicroalgal extract by providing water containing microalgae, providingan amount of flocculant sufficient to form flocs of microalgae whenmixed with water, generating a mixture of the water and flocculant,wherein the mixture is acidic, and separating the floes from themixture, thereby preparing a crude microalgal extract.

The present disclosure further relates to a method of preparing amicroalgal lipid extract by providing water containing microalgae,providing an amount of flocculant sufficient to form floes of microalgaewhen mixed with water, generating a mixture of the water and flocculantunder conditions sufficient to form flocs of microalgae, separating theflocs from the mixture, thereby harvesting the microalgae, andextracting lipid from the microalgae, thereby preparing a microalgallipid extract.

Embodiments of these methods are as described above for the method ofharvesting of microalgae.

EXAMPLES Example 1: Indoor Cultivation 1.1 Purification Process

Nannochloropsis sp. was collected in nature and then purified via asimple plating method, by which a single colony with only one microalgaestrain in it is formed. Here is an example of preparing 1 Liter media tomake agar plate. First 100 ml of each stock solution was prepared asfollows:

TABLE 1 Stock Solutions Stock solution (100 ml) Mass (g) MgSO₄•7H₂O 24.4KCl 6 Urea 2.5 CaCl₂•2H₂O 3 KH₂PO₄•H₂O 0.35 NaHCO₃ 10

22 g of NaCl and 15 g of Agar powder were added into 800 ml di H2O, theneach stock solution was added as follows. Note that final concentrationin the following table is calculated based on each compound in 1 Litermedia

TABLE 2 Agar Plate Media Final concentration Compound Volume (g/L) NaCl/ 2 MgSO₄•7H₂O 10 ml 2.44 KCl 10 ml 0.6 Urea 10 ml 0.25 CaCl₂•2H₂O 10 ml0.3 KH₂PO₄•H₂O 10 ml 0.035 NaHCO₃  2 ml 0.2 P-IV solution  1 ml Pleaserefer to Table 3 Agar / 15

P-IV solution was prepared as follows. Note that the concentration isfinal to 1 L media

TABLE 3 P-IV Solution Stock solution Final concentration Compoundconcentration (g/L) (mg/L) Na₂EDTA•2H₂O 0.75 0.75 FeCl₃•6H₂O 0.097 0.097H₃BO₃ 0.5 0.5 MnSO₄ 1.0 1.0 ZnSO₄ 0.05 0.05 CoCl₂•6H₂O 0.02 0.02Na₂MoO₄•2H₂O 0.1 0.1

di H2O was added up to 1 L, and then pH was adjusted to 7 using HCl orNaOH. The solution was autoclaved at 115 Celsius degree for 30 minutes.100 ml of each vitamin stock solution was prepared as follows. Note thatfinal concentration is calculated on each compound in 1 Liter media.

TABLE 4 Vitamin Stock Solutions Final concentration Stock solution (100ml) Mass (g) (mg/L) Vitamin B1 0.0335 0.335 Vitamin B7 0.0025 0.025Vitamin B12 0.0135 0.135

Before vitamin was added into the media, the vitamin stock solution wasfiltered with a Millipore filter bottle (Millipore Stericup-GP tilterbottle 1000/1000 ml with 0.22 μm polyethersulfone membrane Express pusPER). The media was cooled down to 50-70 Celsius degrees, and 1 ml ofeach axenic vitamin stock solution was added as indicated in Table 4during constant shaking. The media was transferred to petri-dishes, andthen allowed to condense to solid state. An inoculation loop was dippedin the microalgae solution, and a non-overlapped Z-shaped line wasgently drawn on the agar culture. The petri-dishes were closed withlids, and then flipped. The dishes were kept in the incubator at 27Celsius degree. A single colony was picked with inoculation loop andtransferred into cultivation beaker. The composition of media used inthe beaker will be discussed in the next section. When algae accumulatedin the beaker, the above protocol was repeated. After three times, asingle strain was obtained.

1.2 Strain Preservation

After the single strain was collected, it was transferred onto a cleanagar petri-dish (please check above section for method of preparation).The lids were closed, the plates were flipped, and saved at 4 Celsiusdegree.

Under such conditions, algae can typically be stored for half a year.Every three months, the algae from the storage (plates with algae storedat 4 Celsius degree) was plated to activate algae from its hibernation(process in which algae grow and degenerate at a very slow rate). Thisprocess awakens the cells and this batch of algae and their lineagecould maintain its growth and nutritional productivity afterwards.

Plate storage is very susceptible to contamination. Possiblecontaminants include a variety of strains of microalgae, cyanobacteria,bacteria, phytoplankton and zooplankton. It is necessary to controlcontaminants from all possible sources such as inherent foreign strainsfrom the original culture, any incoming microorganisms which are broughtin from lab operation, contaminants in the air, contaminants which arealive after sterilization process, etc. The heterogeneity will causeearly culture to crash. Therefore, plates were divided into differentbatches and stored on separate layers in the incubators.

For production purpose, plates were produced from different preservedinoculums, in case that one specific inoculum was contaminated initiallywithout being noticed. Strain preservation protocols were heavilyexecuted throughout the whole production period.

1.3 In-Lab Media Preparation

Before scaling up algae from plates, sufficient media was prepared. Hereis an example of preparing 1 Liter media for in-lab culture. First, 100ml of each stock solution was prepared as follows:

TABLE 5 Stock Solutions Stock solution (100 ml) Mass (g) MgSO₄•7H₂O 24.4KCl 6 Urea 2.5 CaCl₂•2H₂O 3 KH₂PO₄•H₂O 0.35 NaHCO₃ 10

30 g of NaCl was added into 800 ml di H2O, then each stock solution wasadded as follows. Note that final concentration in the following tableis calculated based on each compound in 1 Liter media

TABLE 6 Growth Media Final concentration Compound Volume (g/L) NaCl / 30MgSO₄•7H₂O 10 ml 2.44 KCl 10 ml 0.6 Urea 10 ml 0.25 CaCl₂•2H₂O 10 ml 0.3KH₂PO₄•H₂O 10 ml 0.035 NaHCO₃  2 ml 0.2 P-IV solution  1 ml Please referto Table 7

P-IV solution was prepared as follows. Note that the concentration isthe final concentration in 1 L media.

TABLE 7 P-IV Solution Stock solution Final concentration Compoundconcentration (g/L) (mg/L) Na₂EDTA•2H₂O 0.75 0.75 FeCl₃•6H₂O 0.097 0.097H₃BO₃ 0.5 0.5 MnSO₄ 1.0 1.0 ZnSO₄ 0.05 0.05 CoCl₂•6H₂O 0.02 0.02Na₂MoO₄•2H₂O 0.1 0.1

di H2O was added up to 1 L, and then pH was adjusted to 7 using HCl orNaOH. The solution was autoclaved at 115 Celsius degree for 30 minutes.100 ml of each vitamin stock solution was prepared as follows. Note thatfinal concentration is calculated on each compound in 1 Liter media.

TABLE 8 Vitamin Stock Solutions Final concentration Stock solution (100ml) Mass (g) (mg/L) Vitamin B1 0.0335 0.335 Vitamin B7 0.0025 0.025Vitamin B12 0.0135 0.135

Before vitamin was added into the media, the vitamin stock solution wasfiltered with a Millipore filter bottle (Stericup-GP filter bottle1000/1000 ml Express pus PER) with 0.22 μm membrane. During the in-labcultivation, media was not stored. Fresh media was prepared as needed.

1.4 Lab Scale-Up

Algae was captured from the preserved plates with inoculation loops andthen transferred into 125 ml beakers (Nalgene™ Single-Use PETGErlenmeyer Flasks with Plain Bottom: Sterile with Nalgene™ Vented HDPEClosures for Sterile Single Use Erlenmeyer Flasks). Media was added tothe beaker until volume reached 50 ml.

These beakers were stored in an incubator where temperature was kept at22-24 Celsius degree and spin rate was set at 150 rpm. The lightintensity inside incubator was set at 35-45 u mol/m2/s, and a mixedcombination of CO2 (1-2% v/v according to pH and cell density in themedia) and air at a flow rate of 0.1 m3/hr was constantly pumped intothe incubator.

After about a week when optical density at 682 nm in the media reached0.1, culture was transferred from 125 ml beakers to 500 ml beakers(Nalgene™ Single-Use PETG Erlenmeyer Flasks with Plain Bottom: Sterilewith Nalgene™ Vented HDPE Closures for Sterile Single Use ErlenmeyerFlasks) and the large beakers were stored in another incubator whichremained at the same condition.

After a week, algae was transferred from 500 ml beakers to 5 L glassbottles. Each glass bottle contained 4 L of culture media. These glassbottles were kept on an open bench with a mixed combination of CO2 (1-2%v/v according to pH and cell density in the media) and air at a flowrate of 0.75 L/min was constantly pumped in. FIG. 1 shows the setup ofair pump and filtration design. The difference between 5 L glass bottlesand smaller beakers in the incubator was that in the glass bottle, airwas directly pumped through the rubber stopper and two connected filters(MTGR05000|Aervent®-50 Filter Unit 0.2 m hydrophobic ⅛ in. HB/HB), whilein the beakers air was firstly pumped into the incubator chamber andthen diffused into the media through the vented cap. The glass bottleswere manually shaken three times a days, 8 am, 12 pm, 5 pm.

After the optical density at 682 nm of algae in the 5 L glass bottlesreached 0.3, algae was transferred into 240 L plate photo-bioreactor.These lined photo-bioreactors were made up of glass containers and steelframes attached with mobile wheels on the ground. Light panels whichconstantly provided light at an intensity of 54-65 mol/m2/s were hungbetween each photo-bioreactor. The culture media in the 2 m*0.2 m*0.6 mphoto-bioreactors remained at a level of 0.5 m out of total height whichequaled 0.6 m. A mixed combination of CO2 (1-2% v/v according to pH andcell density in the media) and air was pumped into each photo-bioreactorat a flow rate of 0.4 m3/hr. Room temperature was kept at 22-24 Celsiusdegree.

Example 2: Analytic Measurement 2.1 Cell Density Measurement

200 ul algae media was sampled each day for cell density measurement.Samples were loaded onto 96 well plates and absorbance was measured at682 nm via microplate reader (Molecular Device SpectraMax M2e).

The following relationship was used to evaluate the biomass of ourproduction strain from the optical density measurement

Dry Cell Weight−OD ₆₈₂*0.687

2.2 Fatty Acid Measurement

The protocol was as follows:

-   -   a. 0.1 gram (the figure is accounted to three decimal places) of        algae dry powder was weighed into 50 ml centrifuge tubes, and 21        ml of 2:1 chloroform(v)/methanol(v) solution was added. This was        shaken on a vortex machine at a rate of 200 r/min for 12 hours.    -   b. The centrifuge tubes were kept stable until the mixture        separated into multi-layers. The supernatant was extracted on        the upper layer and transferred into a glass bottle.    -   c. 21 ml 2:1 chloroform(v)/methanol(v) solution was added to the        unfinished centrifuge tubes. They were shaken on a vortex        machine at a rate of 200 r/min for 3 hours.    -   d. Step b was repeated for the centrifuge tubes.    -   e. All the supernatants were collected in the glass bottle.    -   f. They were then dried with nitrogen gas. 10 ml 5% (v/v) H₂SO₄        methanol solution was added and the bottles were placed in water        bath at 100 degrees Celsius for 1 hour.    -   g. The liquid was allowed to cool down. It was mixed with 2 ml        hexane and certain amount of di-H₂O. The supernatants were        extracted with a syringe and filtered with 0.22 um organic        membrane. The filtrate was loaded on gas chromatography (Agilent        7890B) with FID detector which works at a temperature of 280        degrees Celsius.    -   h. A programmable heater was used from 35° C. (5 min) to 160° C.        (0 min) at a rate of 15° C./min, and then from 160° C. (0 min)        to 280° C. at a rate of 3° C./min. Sample volume was 0.1 ul.

Below is an analysis measured for total fats, total fatty acids, EPA,and polyunsaturated fatty acids.

TABLE 9 Total EPA/ PUFA/ Fats/Algae Total Total dry weight fatty Fattyby GC acids acids Measure Date Algae powder (g/100 g) (%) (%) 2015 Dec.14 Nannochloropsis. sp 21.42 32.38 41.19 2015 Dec. 14 Nannochloropsis.sp 17.90 35.18 42.76 2015 Dec. 30 Nannochloropsis. sp 18.44 42.03 46.85

Example 3: Outdoor Cultivation 3.1 Media Preparation

During outdoor cultivation, unpolluted sea water was directly used andpumped from ocean as the media which may significantly reduce costs.Fresh water was firstly used to adjust the salinity to 22 (NaCl/media=22g/L). Then each component was added (powder was directly poured intoponds) as follows into the media. Any other component was usually notadded into sea water except urea and NaH₂PO₄.2H₂O since natural formulaof sea water was used.

TABLE 9 Final concentration Compound (g/L) Urea 0.25 NaH₂PO₄•2H₂O0.00572

3.2 Pond Design

Open raceway ponds were used to scale up microalgae from the indoorplate photo-bioreactors when OD682 in the bioreactors reached 0.4. Toconstruct a pond, a 0.5 m raceway-shaped trench was first dug on thesoil ground then covered with HDPE films. Several films were welded tocompletely cover the pond and the middle ridge without any leakage.

A paddle wheel connected to an electric motor was set between the ridgeand the siding of pond. The paddle wheel was constantly rotating so thatwater and gas was kept circulating in the pond. Gas tubing was set up onselected spots to the bottom of pond. Industrial flue gas which maycontain 12-14% (v/v) CO2 was pumped directly into water. Water level inthe pond was kept at 30 cm and pH in the water at 7.

3.3 Harvest

For production ponds, all the microalgae was harvested on a weeklybasis. All the liquid in the pond was firstly directed/pumped into aharvest pond (usually the harvest pond is much deeper than productionpond so it can hold liquid from several production ponds).

Harvest ponds can be designed similar to production ponds with the onlydifference being that they are much deeper, e.g. 1.2 m depth. Whenliquid from production ponds flowed into the harvest ponds, the flue gasswitch which control in-flow gas to the ponds was turned to maximal sothat pH in the ponds would drop below 6. Chitosan stock solution (1%chitosan to the biomass contained in the ponds) was then added asharmless flocculant to induce auto-precipitation. Under acidic solution,it causes microalgae in the water to flocculate.

For example, if 1000 metric tons of liquid were in the harvest ponds andOD682 in it read 0.6, the biomass contained in the ponds was0.6*0.687*1000*1000=412.2 kg. Therefore 412.2 kg*1%=4.12 kg chitosan wasadded in the pond.

Chitosan stock solution was prepared by: Adding 0.1 g chitosan powderinto 10 ml 0.1 M HCl solution then filling with water to 100 ml to reacha final concentration of chitosan in the stock solution equivalent to 1g/L.

After flocculation, most of the water was directly drained for recycleuse and then only leaving around 10% of the flocs plus remaining waterin the harvest ponds. The siding of harvest ponds was then washed inorder to collect all the flocs which had stuck to the siding of theponds. These flocs and remaining water were pumped into horizontalcentrifuge. Centrifuging them at a separation coefficient of 2100 easilyseparated the microalgae slurry (over 10% dry algae weight) from theremaining water.

${{Seperation}{coefficient}{RCF}} = {\frac{F_{cf}}{G} = {\frac{m\omega^{2}r}{mg} = \frac{\omega^{2}r}{g}}}$

Pressure filtration was also used to separate microalgae flocculantsfrom remaining water using 500 meshes membrane (30 um pore size).Microalgae slurry was collected through filtration. The microalgaeslurry was fed into spray dryer and dried.

1. A method for harvesting microalgae, the method comprising providingwater comprising microalgae, wherein the water has a pH between about6.0 and about 10.0 and has a salinity between about 0.01% and about 5%;providing flocculant in an amount sufficient to form flocs of microalgaewhen mixed with the water; generating a mixture of the water and theflocculant, wherein the mixture is acidic; and separating the flocs fromthe mixture, thereby harvesting the microalgae.
 2. The method of claim1, wherein the water has a pH between about 8.0 and about 9.0.
 3. Themethod of claim 1, wherein the water has a salinity of about 2.2%. 4.The method of claim 1, wherein the water comprises sodium hydrogencarbonate at a concentration between about 0.01 g/L and about 0.5 g/L.5. The method of claim 4, wherein the water comprises sodium hydrogencarbonate at a concentration between about 0.1 g/L and about 0.2 g/L. 6.The method of claim 1, wherein the water is at a temperature betweenabout 10° C. and about 38° C.
 7. The method of claim 1, wherein theflocculant is chitosan.
 8. The method of claim 1, wherein the flocculantis a polysaccharide that forms cationic polyelectrolytes to precipitatethe microalgae.
 9. The method of claim 7, wherein the chitosan isprovided at a concentration between about 0.08% and about 3.5% of theweight of the microalgae in the mixture.
 10. The method of claim 7,wherein the chitosan is provided at about 2% of the weight of themicroalgae in the mixture.
 11. The method of claim 1, wherein the methodcomprises adding flue gas or wherein the water is mixed with flue gas.12. The method of claim 11, wherein the flue gas comprises carbondioxide.
 13. The method of claim 11, wherein the flue gas has less than5 ppm of sulfur oxide.
 14. The method of claim 11, wherein the flue gascomprises carbon dioxide at a concentration between about 4% and about20%.
 15. The method of claim 11, wherein the flue gas comprises carbondioxide at a concentration between about 10% and about 14%.
 16. Themethod of claim 1, wherein the mixture has a pH between about 4.5 andabout 6.5.
 17. The method of claim 16, wherein the mixture has a pH ofabout 5.5.
 18. The method of claim 1, wherein the microalgae can produceeicosapentaenoic acid (EPA) at temperatures above 32° C.
 19. The methodof claim 1, wherein the microalgae is Nannochloropsis.
 20. A method forpreparing a crude microalgal extract, the method comprising providingwater comprising microalgae, wherein the water has a pH between about6.0 and about 10.0 and has a salinity between about 0.01% and about 5%;providing flocculant in an amount sufficient to form flocs of microalgaewhen mixed with the water; generating a mixture of the water and theflocculant, wherein the mixture is acidic; and separating the flocs fromthe mixture, thereby preparing a crude microalgal extract.
 21. A methodfor preparing a microalgal lipid extract, the method comprisingproviding water comprising microalgae, wherein the water has a pHbetween about 6.0 and about 10.0 and has a salinity between about 0.01%and about 5%; providing flocculant in an amount sufficient to form flocsof microalgae when mixed with the water; generating a mixture of thewater and the flocculant, wherein the mixture is acidic; separating theflocs from the mixture, thereby harvesting the microalgae; andextracting lipid from the microalgae, thereby preparing a microalgallipid extract.